Assay

ABSTRACT

The present invention relates to a method of assaying for folate in a folate containing sample, wherein at least some of the folate comprises at least one attached glutamate residue. The method comprises subjecting the sample to hydrolysis to release paraminobenzoic acid, p-aminobenzoyl glutamic acid, or a salt thereof; contacting the released paraminobenzoic acid, p-aminobenzoyl glutamic acid, salt or a diazo derivative thereof, with a binding partner thereof; and directly or indirectly detecting the resulting binding partner: paraminobenzoic acid, binding partner: p-aminobenzoyl glutamic acid, or salt or derivative combination. The invention further relates to a kit usable in the method of the invention.

This invention relates to an assay for folate as well as to kits andapparatus therefor.

Folate, a term used to cover folic acid, dihydrofolate, tetrahydrofolateand methyltetrahydrofolate, is a coenzyme for the synthesis of thymidinemonophosphate and thus of DNA, and is often referred to as being part ofthe vitamin B complex. It has been widely reported that low levels offolate or a disturbance in folate metabolism is involved in thepathogenesis of several disease states including in particularmegaloblastic anaemia, neural tube defects, cancers and cardiovasculardisease. Folate shortage in maternity has also been linked to childhoodleukaemia. Accordingly the accurate assessment of folate is clinicallyimportant in disease and risk evaluation and monitoring.

Assessment of folate however is complicated by the fact that it occursin a multiplicity of forms varying for example in pteridine ringstructure and the number of glutamate residues and in the distributionof the different variants as the distribution is different for folate inserum and in erythrocytes. Indeed there are currently two widely usedfolate assays—one for serum and the other for whole blood. However forwhole blood folate there are large variances in the interassay andintraassay results from different laboratories. As erythrocyte folategives a measure of the long term status of the patient and is lessinfluenced by food intake than is serum folate, it is generallypreferred to assay for erythrocyte folate; however in view of theproblems with reliably assaying erythrocyte folate some laboratories areproposing to revert to measuring serum folate instead.

It has been proposed to eliminate the problems arising from theheterogenicity of folate by subjecting the assay sample to prolongedboiling (about six hours) in half-concentrated hydrochloric acidfollowed by chromatographic separation and subsequent assessment (e.g.by mass spectroscopy) of a folate fragment that is common to allvariants, namely para-amino benzoic acid (PABA). See for example Anal.Biochem. 283: 266-275 (2000). The effect of this harsh treatment andseparation is to cleave the pteridine ring and strip off all of theattached glycine residues from all variants of folate to leave PABA.Such a technique however is not feasible for routine clinical use, e.g.in diagnostic laboratories or at the point of care. This is particularlydue to the nature of the chromatographic separation step, which is timeconsuming and ill suited to high throughput or multiple-parallelanalysis.

There is thus an ongoing need for a reliable and viable assay for folatein biological specimens.

We have now found that such an assay may be based on detection of PABAwithout requiring its chromatographic separation from the sample andwithout requiring harsh and user-unfriendly or impractical reactionconditions.

Thus viewed from one aspect the invention provides a method of assayingfor folate in a folate containing sample, said method comprising:

-   -   subjecting said sample to hydrolysis to release paraminobenzoic        acid, p-aminobenzoyl glutamic acid (PABA-glu), or a salt        thereof; contacting the released paraminobenzoic acid, PABA-glu        or salt, or a diazo derivative thereof, with a binding partner        therefor; and directly or indirectly detecting the resulting        binding partner:paraminobenzoic acid, binding partner:PABA-glu,        or salt or derivative combination.

Preferably the invention provides a method of assaying for folate in afolate containing sample, said method comprising:

subjecting said sample to hydrolysis to release paraminobenzoic acid ora salt thereof; contacting the released paraminobenzoic acid or salt ora diazo derivative thereof with a binding partner therefor; and directlyor indirectly detecting the resulting binding partner:paraminobenzoicacid or salt or derivative combination.

The sample assayed according to the method of the invention may be anyfolate-containing sample, but especially preferably is blood or derivedfrom blood, e.g. concentrated red blood cells or serum, more especiallyconcentrated (and if desired washed) red blood cells (RBC). With an RBCsample, if desired the cells may be lysed and protein therefromdenatured before the hydrolysis; optionally and preferably however thehydrolysis treatment itself will cause cell lysis and proteindenaturing.

In the assay method of the invention, the binding partner may be insolution or it may be immobilized on a macrostructure, e.g. a solid,liquid or gel particle, or a substrate surface, for example a sheet,rod, tube, fibre, mesh, web, etc. The binding partner:PABA, bindingpartner:PABA-glu, etc combination may be directly detectable, e.g. byvirtue of characteristic radiation emission or absorption, or enzymaticactivity. Alternatively it may be indirectly detectable, e.g. by virtueof the ability of the binding partner to bind to a competitive substancewhich produces a directly detectable combination with the bindingpartner.

In one preferred aspect, the binding partner is an antibody or antibodyanalog capable of binding PABA, PABA-glu, or a PABA derivative, etc,e.g. an antibody, an antibody fragment, a single chain antibody orantibody fragment, an oligopeptide, an oligonucleotide or a smallorganic molecule. Using an appropriate antigen, e.g. PABA, PABA-glu, ora PABA (or PABA-glu) conjugate or derivative, such binding partners maybe selected using conventional techniques, e.g. in vivo antibodygeneration, library techniques such as phage display, combinatorialchemical techniques and computer-aided molecular design.

In a particularly preferred embodiment however the binding partner is anaromatic tertiary amine or phenol or phenol derivative capable ofcoupling to para diazo benzoic acid (PDBA) or paradiazobenzoyl glutamicacid (PDBA) to form a diazo compound having a characteristic lightabsorption or emission. (Light used here includes radiation outside thevisible wavelength range, e.g IR and UV, in particular near IR).

In this embodiment, the released PABA or PABA-glu is converted to PDBA,or PDBA-glu respectively e.g. by reaction with nitrite, preferablybefore being contacted with the binding partner.

Where antibody generation is to be used to select the binding partner,it is preferred to use as the antigen a PABA or PABA-glu substituted atthe 2 or 3 position by a group covalently bound to an antigenicmacromolecular carrier, e.g. a protein such as tetanus toxoid, keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA), or other proteinscommonly used for this purpose. One preferred starting point forproducing such antigens is hydroxy and/or nitro substituted PABA orPABA-glu derivatives, such as for example 2-hydroxy-4-amino-benzoicacid, 2-hydroxy-4-nitro-benzoic acid, 3-hydroxy-4-amino-benzoic acid,3-hydroxy-4-nitro-benzoic acid, or their monoglutamyl amides etc. The2-hydroxy group may readily be reacted with a coupling agent, preferablyafter activation, e.g. tosylation. During the coupling reaction, the4-amino group (and in some cases also the carboxyl group or groups) maydesirably be protected. Conventional protecting groups, e.g. Fmoc, Bocor acetamide for the amine group, and ester formation for the carboxylicacid group or groups, may be used in this regard.

Alternatively, coupling groups or functional groups reactive withcoupling agents may be introduced at the 2 or 3 positions of PABA orPABA-glu using conventional chemical techniques. Where it is desired tointroduce a group at the 2 position, it may be preferred to use paranitro benzoic acid or the monoglutamyl amide thereof and to reduce thenitro group to an amine group (e.g. with Li/H₂) following introductionof the 2-substituent.

The group linking the PABA or PABA-glu to the carrier preferablyprovides a 1 to 50 atom bridge linking the two, more preferably a 5 to20 atom bridge. The backbone atoms of the bridge may or may not be partof an inflexible structure (e.g. an aromatic ring or an aliphatic cage),e.g. to secure correct orientation of the PABA or PABA-glu residue.

Coupling of the 2 or 3 substituted PABA or PABA-glu to the carrier maybe effected in conventional manner, e.g. using thiol terminated2/3-substituents (for example terminated with a Cys residue) anddisulphide or maleimide functionalized carriers.

Less preferably, the antigen for raising anti-PABA or anti-PABA-gluantibodies may be prepared by coupling an antigenic macromolecularcarrier (e.g. a protein) to the carboxy or amino functions of PABAitself or to the amino or one of the carboxy groups of PABA-glu. In thecase of the coupling to the amino function, a Fmoc protectedNHS-activated amino alkanoic acid (e.g. amino propanoic acid) spacer maybe reacted with PABA or PABA-glu, de-protected then coupled to a carrierprotein. Alternatively, a protein coupled to an aldehyde-group carryingspacer may be condensed with the amino group of PABA or PABA-glu toyield an antigen. A further alternative is to react an isothiocyanateactivated aminoalkanoic acid spacer with PABA or PABA-glu or to reactisothiocyanate-activated PABA or PABA-glu with an amino alkanoic acidspacer, optionally after first coupling the spacer to a carrier protein.

The binding partner is preferably substrate bound, e.g. to a porous web(e.g. nitrocellulose) or to polymeric beads, especially preferablymagnetically collectable beads (e.g. such as those available from DynalAS, Oslo, Norway). With an immobilized binding partner, it is possibleto wash the substrate after it has been incubated with the PABA orPABA-glu solution and thus remove other materials in the sample thatmight interfere with determination of the PABA:binding partner orPABA-glu:binding partner combination. This is particularly desirablewhere the sample contains haemoglobin and the binding partner forms anazo compound on reaction with PDBA or PDBA-glu. Standard techniques forcoupling the binding partner to the substrate can be used.

In the performance of the assay of the invention, the conversion offolate to PABA is conveniently effected using a strongly acidic medium,eg 6M hydrochloric acid for 6 hours at 110° C. or by vapour phasehydrochloric acid hydrolysis in sealed containers (eg 1 hour at 150°C.), or 4N methanesulphonic acid. Desirably hydrolysis is effected usinga metal catalyst, e.g a transition metal or compound thereof, inparticular platinum or a platinum compound, in acidic solution, e.g.strong (eg ≧4M, preferably ≧5M, especially ≧6M) hydrochloric acid,optionally after an initial incubation with one or more protease.

In an alternative, highly preferred embodiment, acid hydrolysis offolate to PABA is effected under microwave irradiation. This can bringthe time required for hydrolysis down by a factor of 5 or more, andpossibly even more than 25.

In a further, highly preferred embodiment, oxidation with agents such ashydrogen peroxide, and/or treatment using a reducing agent such assodium borohydride, can be employed to greatly increase thesusceptibility of various folates to acidic hydrolysis by conversion ofthe various folates from the sample into acid sensitive derivatives.These derivatives are then rapidly degradable into PABA or PABA-glu.Preferably, a reduction and an oxidation method are used consecutively.Such a method may comprise, for example, treatment of a sample withsodium borohydride, followed by oxidation with hydrogen peroxide andpotassium permanganate and finally lowering of the pH to around pH1,whereby to convert the various folates into acid susceptible folatederivatives and cause rapid hydrolysis of most, or most preferably all,of folate derivatives to a uniform PABA or PABA-glu product foranalysis.

In addition, folates may be hydrolysed by oxidative photolysis,especially in the presence of photosensitising agents such asriboflavin. Such a method forms a further highly preferred embodimentand may employ visible, infra-red, or particularly ultra-violet light.The major products of such photolysis are typically PABA, PABA-glu andpteridine-6-carboxylic acid (PCA). Any of these products, and preferablyany two or all three products may be determined as described above or byanalogous methods, such as immunoassay of PCA.

Oxidative photolysis of folate may be performed by irradiating thesample with a high intensity light source, typically for >5 minutes ator around room temperature. The effectiveness of the photolysis dependsprincipally upon the presence of dissolved oxygen, the irradiation time,the intensity of the light source and the temperature and pH in thesolution. Certain additives like riboflavin work as photo sensitizersand dramatically increase the yield of the photolysis products. The useof such photosensitizers is thus highly preferred. Low pH (i.e. pH 1-5,especially pH 2-4, e.g. pH=3) is preferred, but is not essential. It canbe carried out to completion even at a pH between 6-9. UV-light (e.g.wavelength around 350 nm or shorter) is preferred for rapid oxidationbut is not essential since oxidation occurs quite rapidly, even whenusing visible wavelengths (e.g. >500 nm).

Oxidative photolysis can be performed directly on lysed samples or onsamples pretreated with enzymes. The different forms of folate are alldecomposed into PABA, PABA-glu and pteridine-6-carboxylic acid as themain products. Although all these species can be determinedimmunologically using antibodies with the necessary specificity, thedetection of PABA or PABA-glu is preferred because the danger ofcross-reactivity of the antibody against endogenous compounds isminimized. PABA and PABA-glutamic acid can be determined by using thefluorescent azo-technique described supra.

The acid hydrolysis may also be made more user-friendly by initialproteolysis, eg using enzymes such as pepsin, trypsin, chymotrypsin andcarboxypeptidase A either alone or in combinations of two or moreoptionally together with further enzymes such as amylase and conjugase.The folate forms released can then be converted to PABA by acidhydrolysis as described above or by enzyme or metal catalyseddegradations. This combination of techniques can result in liberation ofPABA from folate-containing samples in a matter of minutes.

Any of the mild pteridine ring decomposition methods, or any combinationthereof, which are described supra as providing PABA, may be simplymodified to generate PABA-glu. Specifically, incubation of a blood orblood derived sample for a short period (e.g 10 min to 6 hours,preferably 30 min to 3 hours, most preferably 1-2 hours) at aroundphysiological temperature will typically result in the removal of allbut the terminal glutamate residue from the various folates by theaction of naturally occurring conjugase(s). Alternatively, or inaddition, an enzyme or enzyme cocktail comprising components such asconjugases, proteases and α-amylases may be used. In a preferredexample, conjugases, proteases and α-amylases are used as a tri-enzymemixture to accelerate the removal of the glutamate residues. Again theseenzymes result in the removal of all but the final, terminal, glutamicacid residue.

By this method, followed by pteridine ring decomposition as describedabove, the various folates of a sample are converted to a single species(PABA-glu). As described supra, this uniform PABA-glu product may thenbe analysed by the methods used to detect PABA, or simple variantsthereof. The formation and analysis of PABA-glu is thus equallyeffective in reflecting the total folate content as is the analysis ofPABA formed under conditions which would also remove the glutamic acidresidues.

Should the final glutamic acid be desirably removed under the mildconditions preferred in the present invention then the PABA-glu or itsderivatives may be treated with an enzyme such as carboxy peptidase G2under appropriate conditions. Alternatively, this or equivalent enzymesmay be included in the enzymic treatment cocktail described for removalof the other glutamic acid residues, by which method, in combinationwith pteridine ring decomposition, PABA will be generated.

Following the acid hydrolysis to yield PABA or PABA-glu, if the bindingpartner is pH sensitive the pH of the sample may be adjusted (e.g. byaddition of a base such as sodium hydroxide or a buffer), eg to a valuecloser to pH 7.

Where the binding partner is immobilized on a substrate, the sample isthen brought into contact with the substrate and incubated to allowPABA:binding partner or PABA-glu:binding partner, binding to occur.Desirably the substrate is then rinsed to remove unbound material beforethe PABA:binding partner or PABA-glu:binding partner combination isassessed. In this format, it is preferred to use a second, labelledbinding partner which binds either to the PABA/PABA-glu:binding partnercomplex or to the immobilized binding partner which has not bound toPABA/PABA-glu. Detection of the label thus gives a direct or indirectvalue for the PABA or PABA-glu concentration. The labels used may forexample be fluorophores, chromophores, radiolabels, etc.

Where the binding partner is an aromatic amine or phenol, PABA is firstconverted to PDBA or PABA-glu to PDBA-glu, e.g. by reaction with sodiumnitrite and hydrochloric acid. The PDBA or PDBA-glu containing sample isthen contacted with the aromatic amine or phenol which may or may not besubstrate bound. The aromatic amine or phenol must have an unsubstitutedring position ortho or para to the amino or hydroxy group but may besubstituted at other ring positions or at the amine nitrogen and, asdescribed below, such substituents may be used to select the desiredcolour or fluorescence wavelength of the azo compound formed by reactionwith PDBA or PABA-glu as well as to couple the amine or phenol to asubstrate. Where the amine or phenol is substrate bound, afterincubation with the PDBA or PDBA-glu containing sample, the substratemay be rinsed to remove unbound material and the azo compoundconcentration can then be determined directly by spectrometric methods.Where the amine or phenol is not substrate bound, the concentration ofthe azo compound can again be determined directly by spectrometricmethods. However as the sample will contain heme degradation products,in this case the amine or phenol is preferably selected so that the azocompound is fluorescent or has a characteristic absorption at awavelength at which the “background” from the heme degradation productsis relatively small.

Hydroxyl and amino groups, especially if they are ortho- or para- to theazo bond intensify the colour of azo compounds. This is exemplified byp-hydroxyazobenzene which has an absorption maximum at 349 nm (ethanol,ε=26300), and p-dimethylaminoazobenzene which has an absorption maximumat 408 nm (ethanol, ε=27540). Both azo-compounds absorb at longerwavelength and with higher molar absorptivity than azobenzene itselfdoes.

Azo-compounds in which an electron donating substituent on one aromaticring is conjugated with an electron-withdrawing group on the other ringhave especially deep colours. A good example is provided by the group ofazobenzenes where a nitro group is substituted on one ring ortho or parato the azo-linkage and a dialkylamino group is substituted on the otherring—also para to the azo linkage. This is because one resonancecontributor of these compounds has a quinoid structure, a feature oftenassociated with deep colour in azo compounds. An example of such acompound illustrating this characteristic is4-dimethylamino-4′-nitrobenzene which has an absorption maximum at 478nm with ε=33110 in ethanol.

Fluorescence may be expected-generally in molecules that are aromaticand contain multiple-conjugated double bonds with a high degree ofresonance stability. Both classes of substances have delocalizedn-electrons that can be placed in low-lying excited singlet states. Oneway of increasing the fluorescence of azo compounds is to prevent theexcited single state from loosing the energy by intramolecularnonradiative transitions, i.e. transforming the excitation energy intovibrational movements within the molecule. Molecular rigidity lessensthe possibility of competing nonradiative transitions by decreasingvibrations this minimizes intersystem crossing to the triplet state andcollisional heat degradation. This is clearly illustrated by extendingthe alkyl chains of the tertiary amino group in4-dialkylamino-4′-carboxyazobenzene. Many short alkyl chain lengthazobenzenes are practically non-fluorescent, while longer chain (≧C3)azobenzenes are fluorescent. Also, the fluorescence intensity increaseswith the alkyl chain length of the amino group. However, since thisinevitably increases the hydrophobicity of the compounds, the decreasedsolubility of these long chain fluorescent azo-compounds may limit theiruse. Thus 4-di (C₃₋₆alkyl)amino-4′-carboxyazobenzenes are especiallypreferred.

Since reduced flexibility of the alkyl chain is a key element related tothe fluorescence properties, one way of overcoming the solubilityproblem and further “immobilize” the alkyl chains is to use aromaticamines where the alkyl chains form stable ring structures. An example ofsuch a compound is julolidine. This structure, incorporated in theazo-structure with the amine para to the azo bond, greatly reduces lossof excitation energy due to molecular vibration, resulting in increasedfluorescence. Furthermore, adding extra phenyl rings in theazo-compounds, like those formed when naphthalene-derivatives (or higherorder fused phenyl ring compounds) with amino and/or hydroxylsubstituents (naphthol/naphthaleneamine) are reacted with diazoniumgroup of PDBA or PDBA-glu, strengthens the fluorescence.

Generally, the most planar, rigid and sterically uncrowded molecule of aseries of organic compounds is the most fluorescent one. The formationof chelates with metal ions in general also promotes fluorescence bypromoting rigidity and minimizing internal vibrations. For this reason,hydroxyl or substituted or unsubstituted amino groups are preferablyincluded in the amine or phenol at positions meta to the amine orhydroxyl groups. These will be capable of coordinating metal ions.

Such substituents strongly affect fluorescence. A substituent thatdelocalises the n-electrons, such as an —NH₂, —OH, —OCH₃, —F, —NHCH₃ orN(CH₃)₂ group, often enhances fluorescence, while electron-withdrawingsubstituents, e.g. —Cl, —Br, —I, —NHCOCH₃, —NO₂ or —COOH, decrease orcompletely quench the fluorescence.

Additionally, changes in the system pH may also influence fluorescenceif it affects the charge status of the chromophore. Such changes can beexplained by comparing resonance forms of anion and cation structures.

The substitution patterns and conjugated ring systems that enhance theconjugated system and/or lead to quinoid structures will generally giverise to bathochromic shifts resulting in absorption bands in the redpart of the spectrum, possibly different from the absorption bands ofthe heme decomposition products. It is important to realise that thehydrolysis of hemeglobin will produce heme-decomposition products withabsorption characteristics different from native heme, and the majorityof these compounds will absorb in the blue part of the spectrum (and soappear light yellow-brownish in colour). This is obviously a beneficialsituation since the spectral overlap between the heme derivedchromophores and the diazo compound will be greatly reduced compared tothe situation with native heme.

Where the binding partner is neither substrate bound nor an aromaticamine or a phenol, the PABA or PABA-glu containing sample is desirablyfirst incubated with a labelled binding partner and then contacted witha second, substrate bound binding partner which is capable of bindingeither to the PABA/PABA-glu:binding partner complex or to the freebinding partner. The substrate is then desirably rinsed to removedunbound material and the substrate-bound label is then detected giving adirect or indirect value for the PABA or PABA-glu concentration.

Alternatively, an unbound, fluorophore-labelled binding partner may beused with the concentration of PABA or PABA-glu being determined by thechange in fluorescence polarization resulting from the formation ofPABA/PABA-glu:binding partner complexes. Again however the fluorophorepreferably has a characteristic emission wavelength at which hemedegradation products provide minimal background.

Viewed from a further aspect, the invention provides a kit, for use inthe performance of the assay of the invention, said kit comprising:

-   -   a) a folate hydrolysis reagent;    -   b) optionally an enzyme cocktail;    -   c) a PABA, PABA-glu, PDBA or PDBA-glu binding partner;    -   d) optionally a [PABA to PDBA or PABA-glu to PDBA-glu        converting] reagent; and    -   e) optionally, a secondary binding partner.

The invention preferably provides a kit for use in the performance ofthe assay of the invention, said kit comprising:

-   -   a) a folate hydrolysis reagent;    -   b) a PABA or PDBA binding partner;    -   c) optionally a [PABA to PDBA converting] reagent; and    -   d) optionally, a secondary binding partner.

The assay method of the invention is preferably performed together witha homocysteine assay and/or a holo transcobalamin II assay, e.g. asdescribed in U.S. Pat. No. 6,063,581, U.S. Pat. No. 5,631,127, WO00/40973, WO 00/11479 and WO 00/17659.

The invention will now be described further with reference to thefollowing non-limiting Examples:

EXAMPLE 1

Conversion of Folate to PABA

A 200 μL sample of whole blood is mixed with 500 μL of 8.5M hydrochloricacid in borosilicate glass tubes with Teflon-lined screw caps andincubated for 6 hours at 110° C.

EXAMPLE 2

Conversion of PABA to PDBA

The PABA containing composition of Example 1 is cooled to roomtemperature, diluted with 500 μL purified water and loaded onto a C18solid phase extraction cartridge. The colourless eluant is diluted 1:10v/v with water to produce a hydrogen chloride concentration of approx.0.3M. Aqueous sodium nitrite solution (50 mg/ml) is added in a volumeratio of 2:1 (sample:NaNO₂ solution) and the mixture is allowed to reactfor ten minutes at 4° C.

EXAMPLE 3

Conversion of PDBA to Diazo Compound

The PDBA solution of Example 2 is mixed with an aromatic amine or phenolsolution (4 mg/mL in 10 mM aqueous phosphate buffer, 0.15M NaCl, pH 7.4)in a volume ratio of 3:2 (PDBA solution:amine/phenol solution) andallowed to react for 30 minutes. The diazo compound can then be detectedspectrometrically.

In this Example, the following aromatic amines/phenols will typically beused: N,N-di-n-propyl-aminobenzene; N,N-di-n-butyl-aminobenzene;julolidine; phenol; and 2-hydroxynaphthalene.

EXAMPLE 4

Combined Use of Hydrogen Peroxide and pH to Decompose all Folate Speciesinto PABA-Monoglutamic Acid

1. Add 25 μl whole blood and lyse the cells using saponin (add to 100mg/L final concentration) or ascorbic acid (added to 1% ascorbic acid,final concentration). Incubate for 2 hours at 37° C. to allow theconjugase present in the sample to remove all but the terminal glutamateresidue.

2. Remove non-relevant molecules from whole blood using a molecularsieve (e.g. centrifuge concentrators) to leave fairly purefolate/binding protein complexes.

3. Dissociate folate from binding protein using 0.1% (finalconcentration) DTT and boil for 15 minutes or alternatively add DTT and0.15N (final concentration) sodium hydroxide and incubate 2.5 minutes at37° C.

4. Adjust pH to 6.0 and add sodium borohydride (6 mg/ml) and incubate atroom temperature for 10 minutes to reduce folates.

5. Acidify using HCl to adjust pH and destroy the borohydride.

6. Adjust pH to 9.0 and add hydrogen peroxide and potassium permanganate(0.015% H₂O₂ and 0.1% KMNO₄, respectively) to oxidize, followed byexcess hydrogen peroxide (0.3% H₂O₂) to precipitate the excesspermanganate. The precipitated MnO₂ is removed by centrifugation 4000 gfor 10 min.

7. Add catalase (0.1% catalase to a final concentration of ⅓ the volumeof H₂O₂ present) to destroy the hydrogen peroxide.

8. Reduce pH to 1 with HCl and incubate for 2 hours at room temperatureto drive the conversion of folate to PABA-glu.

9. Adjust the acidity of the sample to neutral pH and determine theamount of PABA-glu present either by using a specific antibody or bytransforming PABA-glu into a fluorescent azo-dye as described e.g inExample 3. The amount of PABA-glu is determined from a standard curveand the resulting concentration reflects the total concentration offolate in the sample.

EXAMPLE 5

Accelerated Removal of Non-Terminal Glutamates

As for Example 4, but in step 1, additional conjugases, like γ-Glu-XCarboxypeptidase; EC 3.4.19.9, or a trienzyme mixture composed ofconjugase, protease and α-amylase, are added to help facilitate therelease and transformation of folate and speed up the reactions.

EXAMPLE 6

Combined Use of Hydrogen Peroxide and pH to Decompose all Folate Speciesinto PABA

1. Step 1 as for Examples 4 and 5 but the proteolytic enzymeCarboxypeptidase G2 is used alone or in addition to the enzymesmentioned in Example 5. This further enzyme results in the removal ofall glutamic acid residues from folate, resulting in the production ofPABA rather than PABA-glu.

2. Remove non-relevant molecules from whole blood using a molecularsieve (e.g. centrifuge concentrators) to leave fairly purefolate/binding protein complexes.

3. Dissociate folate from binding protein using 0.1% (finalconcentration) DTT and boil for 15 minutes or alternatively add DTT and0.15N (final concentration) sodium hydroxide and incubate 2.5 minutes at37° C.

4. Adjust pH to 6.0 and add sodium borohydride (6 mg/ml) and incubate atroom temperature for 10 minutes to reduce folates.

5. Acidify using HCl to adjust pH and destroy the borohydride.

6. Adjust pH to 9.0 and add hydrogen peroxide and potassium permanganate(0.015% H₂O₂) and 0.1% KMNO₄, respectively) to oxidize, followed byexcess hydrogen peroxide (0.3% H₂O₂) to precipitate the excesspermanganate. The precipitated MnO₂ is removed by centrifugation 4000 gfor 10 min.

7. Add catalase (0.1% catalase to a final concentration of ⅓ the volumeof H₂O₂ present) to destroy the hydrogen peroxide.

8. Reduce pH to 1 with HCl and incubate for 2 hours at room temperatureto drive the conversion of folate to PABA.

9. Determine the amount of PABA present either by immunodetection usinga specific antibody (after first adjusting the pH to an appropriate pHoptimal for the immunoreaction, e.g. to a pH between 7.0 and 8.5) oralternatively by transforming PABA into a fluorescent azo-dye asdescribed herein e.g. in Example 3. The amount of PABA is determinedfrom a standard curve and the resulting concentration reflects the totalconcentration of folate in the sample.

EXAMPLE 7

Use of Oxidative, Photolysis of Folate Species into PABA Photolysis, butWithout the Use of Endogenous Enzymes

1. Lyse red cells using saponin (add to 100 mg/L final concentration) orascorbic acid (added to 1% ascorbic acid, final concentration) andincubate for 1 hours at 37° C.

2. Identical to step 2, Example 4.

3. Identical to step 3, Example 4.

4. Irradiate the sample with a UV-light source for 10 minutes at roomtemperature. The main photolysis products are PABA, PABA-glu andpteridine-6-carboxylic acid which all reflect the initial totalconcentration of folate in the sample.

5. Determine the amount of PABA or PABA-glu present either byimmunodetection using a specific antibody (after first adjusting the pHto an appropriate pH optimal for the immunoreaction, e.g. between 7.0and 8.5) or alternatively by transforming PABA or PABA-glu into afluorescent azo-dye as described in Example 3. The amount of PABA orPABA-glu in the sample is determined from a standard curve made withappropriate standards covering the concentration range of folate inblood samples.

1. A method of assaying for folate in a folate containing sample,wherein at least some of said folate comprises at least one attachedglutamate residue, said method comprising: subjecting said sample tohydrolysis to release paraminobenzoic acid, p-aminobenzoyl glutamicacid, or a salt thereof, contacting the released paraminobenzoic acid,p-aminobenzoyl glutamic acid, salt, or a diazo derivative thereof, witha binding partner therefor; and directly or indirectly detecting theresulting binding partner: paraminobenzoic acid, binding partner:p-aminobenzoyl glutamic acid, or salt or derivative combination whereinsaid method does not comprise any chromatographic separation steps.
 2. Amethod as claimed in claim 1 wherein said sample is a blood derivedsample.
 3. A method as claimed in claim 1 wherein said binding partneris selected from an antibody, an antibody fragment, a single chainantibody, a single chain antibody fragment, an oligopeptide,anoligonucleotide and a small organic molecule.
 4. A method as claimedin claim 3 wherein said small organic molecule is an aromatic tertiaryamine, phenol or phenol derivative capable of forming a diazo compoundwith paradiazobenzoic acid (PDBA) or paradiazobenzoyl glutamate(PDBA-glu).
 5. A method as claimed in claim 1 wherein said hydrolysiscomprises treating said sample with a metal catalyst under acidicconditions.
 6. A method as claimed in claim 1 wherein said hydrolysiscomprises treating said sample with microwave radiation.
 7. A method asclaimed in claim 1 wherein said hydrolysis comprises treatment with anoxidising agent.
 8. A method as claimed in claim 7 wherein the oxidisingagent is hydrogen peroxide and/or potassium permanganate.
 9. A method asclaimed in claim 1 wherein said hydrolysis comprises treatment with areducing agent.
 10. A method as claimed in claim 9 wherein the reducingagent is sodium borohydride.
 11. A method as claimed in claim 1 whereinsaid hydrolysis comprises oxidative photolysis.
 12. A method as claimedin claim 11 wherein said oxidative photolysis is carried out in thepresence of a photosensitiser.
 13. A method as claimed in claim 1wherein said sample is incubated in the presence of naturally occurringand/or added enzymes whereby to remove all but the terminal glutamateresidue from said folate and wherein the product of the hydrolysis isPABA-glu.
 14. A method as claimed in claim 1 wherein said sample isincubated in the presence of at least one added enzyme, whereby toremove all glutamate residues from said folate, and wherein the productof the hydrolysis is PABA.
 15. A method as claimed in claim 1 whereinsaid binding partner: paraminobenzoic acid, binding partner:p-aminobenzoyl glutamic acid, or salt or derivative combination isdetected directly by absorbence or fluorescence.
 16. A method as claimedin claim 1 wherein said binding partner: paraminobenzoic acid, bindingpartner: p-aminobenzoyl glutamic acid, or salt or derivative combinationis detected indirectly by means of a secondary binding partner.
 17. Akit for use in the performance of the assay of the invention, said kitcomprising: i) a folate hydrolysis reagent; and ii) a PABA, PABA-glu,PDBA or PDBA-glu binding partner.
 18. A kit as claimed in claim 17additionally comprising an enzyme or enzyme cocktail.
 19. A kit asclaimed in claim 17 additionally comprising a PABA to PDBA or PABA-gluto PDBA-glu converting reagent.
 20. A kit as claimed in claim 17additionally comprising a secondary binding partner.